1,720,965 research outputs found
Heparin-induced structural modifications and oxidative cleavage of human serum albumin in the absence and presence of glucose.
No full textBoth unfractionated and fractionated, low-molecular-mass heparins were tested on human serum albumin in the absence and presence of glucose at concentrations similar to those frequently found in diabetic hyperglycaemic patients, to ascertain whether heparin and glucose interfered with each other in affecting the conformation of albumin. Reproducible results were obtained with both heparins when used at equal masses, but not when used at equal molar concentrations, suggesting a crucial role of the amount of the saccharide units in determining the observed effects. Spectroscopic studies showed that the binding sites of glucose and heparin on albumin do not overlap and that changes in protein structure depend on complex and mutual interference of glucose and heparin with the protein, although the effects of heparin in modifying the chromophore environment and increasing the ordered structure of the protein also prevailed in the presence of glucose. Heparin binding to albumin rapidly gave rise to oxidative reactions, which were responsible for the increase in the carbonyl content of the protein together with its higher susceptibility to tryptic digestion. Glucose enhanced and prolonged the production of heparin-induced oxidants. Oxidation caused peptide bond cleavage at Lys323 in the primary structure of albumin, yielding two large fragments of 27.5 kDa and 35 kDa which aggregated to form disulphide-linked homodimers visible in SDS/PAGE as two new bands of 54 kDa and 74 kDa, respectively. This was accompanied with a reduction in Val, Glu, and Gly residues, only partially counterbalanced by an increase in Thr and Ser residues. While only a small percentage of albumin molecules underwent fragmentation in the presence of heparin with glucose, albumin turned out to display in an even higher proportion structural modifications consistent with a higher degree of ordered structure. The mechanism(s) underlying this heparin-driven effect and possible physiopathological implications in vivo are discussed
A heat shock protein70 fusion protein with alpha(1)-antitrypsin in plasma of Type 1 diabetic subjects
No full textThe recent observation that heat shock proteins (HSPs), mostly glucose regulated protein94 (Grp94) and HSP70, are present in plasma of Type 1 diabetic subjects as complexes with immunoglobulins, prompted us to investigate the nature and extent of this association, whether it represents HSP-induced activation of the immune system. Two complementary affinity chromatography procedures followed by immunoprecipitation and immunoblot analyses of HSP-enriched, plasma-purified peaks, revealed that HSPs were inextricably linked with IgG in SDS-resistant complexes from which proteins dissociate partially under reducing treatment. HSP70 was found also closely linked with α1-antitrypsin (α1AT) in a single protein having the mass of α1AT but elution characteristics different from those of normal α1AT. Immunoprecipitation with anti-HSP70 antibodies led to co-immunoprecipitation of the α1AT species linked to HSP70, thus confirming fusion of the proteins. The additional finding of circulating antibodies against the HSP70-α1AT protein supported its immunogenic properties with implications for diabetes and its complications
Both a serine-like protease and a protease inhibitory activity are present in circulating immune complexes in type 1 diabetes mellitus.
No full textEffects of intact and reduced reducing termini in heparin on structural and functional modifications of trypsin.
No full textAlbumin contamination of a purified human alpha1-antitrypsin preparation does not affect either structural conformation or the electrophoretic mobility of the inhibitor.
No full textA partially purified preparation of human alpha 1-antitrypsin (alpha 1-AT) shown to be 60% active as an inhibitor of bovine trypsin, was chosen as starting material to investigate the nature and extent of contamination by human serum albumin (HSA) and to see whether or not such a contamination was responsible for both the reduced inhibitory activity and the slower migratory rate of the proteinase inhibitor in SDS-PAGE. Immunoblotting analysis revealed the presence of HSA in the unprocessed preparation of alpha 1-AT which, both in denaturing and non-denaturing PAGEs, had the same mobility as HSA, appearing as a single band of 65 kDa. By submitting the unprocessed alpha 1-AT preparation to affinity chromatography on an Affi-Gel Blue chromatography column, an apparently highly purified and homogeneous form of alpha 1-AT was obtained, as confirmed by measurement of inhibitory activity and densitometric scanning of SDS-PAGE in non-reducing conditions. However, immunoblotting analysis still revealed the presence of HSA in the most active fractions of the inhibitor eluted from the column, and regardless of purification degree, the molecular mass of the inhibitor was always 65 kDa. Treatment with beta-mercaptoethanol led to separation in SDS-PAGE of HSA as a distinct band of about 10 kDa higher than the alpha 1-AT band, which instead maintained the same mobility as in non-reducing conditions. The results indicate that HSA has not been completely removed from alpha 1-AT, and its presence does not affect the electrophoretic mobility of the inhibitor. The possibility that the structural conformation of the alpha 1-AT, rather than contamination with HSA, was responsible for its abnormal slower migratory rate was therefore tested. For this purpose alpha 1-AT preparations of different degrees of purification were submitted to heat treatment to induce a non-inhibitory conformation such as loop-sheet polymerization. Polymerization was followed both by the appearance in SDS- and non-denaturing PAGEs of high molecular weight bands, which were mostly present in less purified preparations of the inhibitor, and by a decrease in inhibitory activity. A higher degree of polymerization with complete loss of inhibitory activity was observed in the unprocessed alpha 1-AT preparation when dissolved in Na-phosphate buffer at acidic pH, and after dialysis. After heat treatment, the purified alpha 1-AT was shown to run faster in the gel and, in both reducing and non-reducing conditions, the calculated mass of the inhibitor was that expected of about 54 kDa. After reducing treatment, high molecular weight polymers in SDS-PAGEs were reduced, strongly suggesting that disulphide bridges are also involved in the polymerization of alpha 1-AT. Results indicate that the mobility of alpha 1-AT in SDS-PAGE is crucially dependent on its structural conformation which dictates the extent of SDS binding. HSA contaminating the alpha 1-AT preparation does not affect the inhibitor conformation although at a higher degree of contamination and in favourable conditions, it does reduce the inhibitor activity
Spin-trapping agent alpha-phenyl N-tert-butylnitrone binds to trypsin and enhances heparin-induced inhibition of amidolytic activity and structural degradation of the enzyme.
No full textThe effects of heparin on trypsin have recently been demonstrated to involve inhibition of catalytic activity and degradation of the enzyme by means of an oxidative mechanism. The possibility that alpha-phenyl N-tert-butylnitrone protects heparin-induced radical formation on trypsin was investigated by measuring amidolytic activity and changes in the structure of trypsin in the presence of heparin with and without alpha-phenyl N-tert-butylnitrone. The results show that alpha-phenyl N-tert-butylnitrone does not only prevent, but it even significantly enhances effects of heparin on the enzyme. This is due to the unique property of alpha-phenyl N-tert-butylnitrone, independently of spin-trapping capacity, to modify the trypsin structure by binding irreversibly to the catalytic triad, at sites distinct from those to which heparin binds
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
- …
